Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von Willebrand factor-induced platelet agglutination.

Abstract

Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the β subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the β subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF A1 domain and GPIbα indicated that Asp62, Arg115, and Lys117 of the β subunit are located near Arg218 and Asp222 of GPIbα, respectively, and that Aspβ70 is in proximity to Gln1391 of the A1 domain. Our results indicate that these charged amino acid residues in the β subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.

• Authors: Yukiyo Yamamoto-Suzuki, Yoshihiko Sakurai, Yoshihiro Fujimura, Masanori Matsumoto, Jiharu Hamako, Tetsuro Kokubo, Hitoshi Kitagawa, Sarkar M A Kawsar, Yuki Fujii, Yasuhiro Ozeki, Fumio Matsushita, Taei Matsui
• Journal: Biochemistry (Biochemistry) Vol. 51 Issue 26 Pg. 5329-38 (Jul 03 2012) ISSN: 1520-4995 [Electronic] United States
• PMID: 22647083 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Crotalid Venoms
• Recombinant Proteins
• Snake Venoms
• von Willebrand Factor
• botrocetin

Topics

• Amino Acid Sequence
• Animals
• Cell Line
• Crotalid Venoms (chemistry, genetics, metabolism)
• Humans
• Molecular Sequence Data
• Platelet Aggregation (drug effects)
• Protein Structure, Secondary
• Recombinant Proteins (chemistry, genetics, metabolism)
• Sequence Homology, Amino Acid
• Snake Venoms (metabolism)
• von Willebrand Factor (pharmacology)